Coenzyme Q (Coenzyme Q: CoQ) is a benzoquinone derivative and is named as a ubiquinone since it widely exists in the living world. A hydroquinone obtained by 2-electron-reducing a ubiquinone is a ubiquinol.
The compound name of the ubiquinone is 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone, and a great number of homologs with n=1 through 12 isoprene units naturally exist, wherein n=10 for higher animals such as humans. In the following description, a ubiquinone of humans, etc., is represented as coenzyme Q-10 and a ubiquinol of humans, etc., is represented as a 2-electron reduced form of coenzyme Q-10, as long as no particular reference is made.
The 2-electron reduced form of coenzyme Q-10 has a strong oxidative effect and is considered to have an antiaging effect such as the prevention of cellular damage caused by active oxygen.
Oxidation stress is considered to be an undesired state for an organism in which the balance between oxidation and antioxidation in the organism is disrupted so as to shift toward oxidation, and it is considered that the ratio of the coenzyme Q-10 and the 2-electron reduced form thereof can be a good marker for oxidation stress since it is considered to reflect the degree of oxidation stress.
Thus, since it is very useful to know the behaviors of the coenzyme Q-10 and the 2-electron reduced form thereof, a method for accurately analyzing these components is desired.
As a conventional analysis method, an ultraviolet absorption method, etc., is provided but it is easily subjected to the influence of a third substance and, therefore, a complicated pretreatment is required.
In recent years, a high-performance liquid chromatography (referred to as a HPLC below) has been widely used as a method that can attain a highly sensitive and accurate analysis. For the detection of coenzyme Q-10, ultraviolet absorption at 275 nm is utilized. However, the sensitivity of the conventional analysis method by means of a HPLC is not enough to detect coenzyme Q-10 in blood plasma.
For this reason, a method of reducing coenzyme Q-10 to a 2-electron reduced form thereof for quantifying the difference between the quantities of the coenzyme Q-10 before and after the reduction is also suggested. However, in this analysis method, it is necessary to perform a pretreatment on a specimen and to twice perform sample injections into a HPLC.
Therefore, the applicants previously suggested a method such that after coenzyme Q-10 and a 2-electron reduced form thereof are separated by a reversed phase separation column 1 (LC-8 produced by SUPELCO), the coenzyme Q-10 is reduced to a 2-electron reduced form thereof using a reduction column 2 (SHISEIDO CQ produced by Shiseido Co., Ltd.) or coulometric electrodes, which reduced forms are measured by an electrochemical detector 3, as shown in FIG. 1 (For example, see Satosi Yamasita and Yorihiro Yamamoto, ANALYTICAL BIOCHEMISTRY, 250, 66-73 (1997)). Herein, in FIG. 1, reference numerals 4, 5, 6, 7, and 8 denote a mobile phase, a pump, a sample injector, a protection column, and an ultraviolet absorption detector, respectively.
The highly sensitive simultaneous measurement of the coenzyme Q-10 and the 2-electron reduced form thereof can be attained using the analysis method described above. One example of an obtained chromatogram is shown in FIG. 2. In FIG. 2, “1” denotes a peak for the coenzyme Q-10 and “2” denotes a peak for the 2-electron reduced form of the coenzyme Q-10.
In this case, since the measurement is influenced by a water-soluble antioxidation substance in the specimen, such as vitamin C and uric acid, etc., a pretreatment is performed for subjecting the specimen to an extraction treatment with methanol/hexane so as to distribute the water-soluble antioxidation substance into a methanol phase and the coenzyme Q-10, etc., into a hexane phase.
However, when pretreatment is performed subjecting the specimen to the extraction treatment with methanol/hexane, the coenzyme Q-10 in the hexane extraction liquid is chemically unstable. After the pretreatment and until the pretreated liquid is injected into a HPLC for analysis, the 2-electron reduced form of coenzyme Q-10 is oxidized at a substantial rate as shown in FIG. 3. It is therefore necessary to perform analysis immediately after the extraction of the specimen. For performing an accurate analysis, since it is essential to perform an extraction process immediately before the injection into the HPLC, it is significantly difficult to treat a large amount of specimen collectively. Herein, each temperature indicates a storage temperature of the hexane extraction liquid in FIG. 3.